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The spleen is a vertebrate organ that has both hematopoietic and immunologic function. The embryonic origins of the spleen are obscure, with most studies describing the earliest rudiment of the spleen as a condensation of mesodermal mesenchyme on the left side of the dorsal mesogastrium. The development of spleen handedness has not been described previously, presumably because of the difficulty in assaying spleen position in the embryo and the lack of early, organ-specific molecular markers. Here we show that expression of the homeobox gene Nkx2-5 serves as a marker for spleen precursor tissue. Pre-splenic tissue is initially located in symmetric domains on both sides of the embryo but, during subsequent development, only the left side goes on to form the mature spleen. Therefore, the final location of the spleen on the left side of the body axis appears to result from preferential development of the spleen precursor cells on the left side of the embryo. Our studies indicate that the spleen and heart become asymmetric via different cellular mechanisms. Nkx2-5 may function locally as part of the laterality cascade, downstream of nodal and Pitx2, or it may direct asymmetric morphogenesis after laterality has been determined.
Fig. 1. Xenopus Nkx2-5 is expressed in the spleen in late embryos and adult frogs.
(A) By RNase protection, XNkx2-5 transcripts are detected abundantly in adult spleen and also in adult heart. (B,C) In situ hybridization indicates that Nkx2-5 transcripts are localized to a small crescent on the dorsal side of the stomach at stage 43, the established position of the developing spleen (Nieuwkoop and Faber, 1994). (A) RNase protection analysis of Nkx2-5 and control EF-1α transcripts in adult tissues. (B) Ventral view of a stage 43 tadpole assayed for Nkx2-5 transcripts. (C) Side view of the embryo in B. g, gut tube; h, heart; k, kidney; li, liver; lu, lung; p, pancreas; sk, skeletal muscle; sp, spleen; st, stomach.
Fig. 2. Xenopus Nkx2-5 transcripts in the abdomen become asymmetrical during development. In addition to the heart and pharyngeal region, Nkx2-5 transcripts are detectable by in situ hybridization, beginning at stage 37/38, in two bilaterally symmetric regions of mesoderm overlying the foregut. Within several hours, Nkx2-5 expression disappears from the right side, while it is maintained on the left side (A,B,E,F,I,J). While asymmetry is being generated, the right and left regions of expression, detected by in situ hybridization, remain disconnected (C,G,K) and no migration of Nkx2-5-expressing cells is detectable in cross-sections through the foregut region (D,H,L). (A-D) Stage 37 embryos; (E-H) stage 39 embryos; (I-L) stage 40 embryos. The embryos in A, E and I are photographed from the left side. B, F and J are right-side views of the embryos in A, E and I, respectively. C, G and K are ventral views and the left-side of the embryo is on the right side of the photographs. D, H and L are transverse sections through the foregut region. The left side of the embryo is on the right side of the photographs. The gut (g), heart (h) and melanocytes (m) are indicated. Arrows mark Nkx2-5 expression in the putative spleen precursors.
Fig. 3. Detection of apoptotic cells in histological sections. Transverse sections through a stage 39 embryo showing apoptotic cells at the level of the eye (A) and the foregut (B). In no case was a difference observed in the level of apoptotic cells between the left and right sides in the foregut between stages 37 and 40. a, apoptotic cells; cg, cement gland; e, eye; m, melanocytes.
Fig. 4. XNkx2-5 expression is maintained in explants of pre-splenic tissue. (A) Tissue explanted from the left side at stage 28. (B) Tissue explanted from the right side at stage 28. All explants were cultured until stage 41, then assayed by in situ hybridization using an XNkx2- 5 probe. Note that explants from both sides of the embryo continue to express Nkx2-5.
Fig. 5. The handedness of foregut looping and XNkx2-5 expression are correlated in UV-treated embryos. (A) Side-view of a stage 40 individual with reversed foregut looping, reversed XNkx2-5 expression and normal heart looping. (B) Lateral view of a stage 40 individual with normal foregut looping, normal XNkx2-5 expression and reversed heart looping. (C) Stage 41 embryo with reversed XNkx2-5 expression and foregut looping. (D) Stage 41 embryo with normal XNkx2-5 expression and normal foregut looping. C and D were photographed at an angle between the ventral and lateral sides so that both the looping foregut and XNkx2-5 expression are visible. Arrows indicate XNkx2-5 expression in the spleen. XNkx2-5 is also expressed in the heart (h) in these embryos, although it is sometimes obscured by the liver (L).
Fig. 6. Bilateral XNkx2-5 expression in UV-treated embryos. In situ hybridization of a UV- treated embryo in which XNkx2-5 expression is maintained on both the right and left sides at stage 41. The embryo is viewed from the ventral side. h, heart; r, right; l, left.
